Transmission spectroscopy is used to perform quantitative analysis of a sample based on the transmission of a light beam through a sample contained by a sample cell. Different frequency components of the light beam are absorbed by components of the sample, whereby a frequency analysis of light transmitted through the sample permits analysis of the sample itself. Dry chemical reagents are dissolved by the sample and react with the analyte of interest to produce a chromaphoric response at certain wavelengths of light ranging from about 450 nanometers (“nm”) to about 950 nm.
Transmission spectroscopy is one method for measuring the concentration of an analyte (e.g., glucose, lactate, fructosamine, hemoglobin A1c, and cholesterol) in a body fluid (e.g., blood, plasma or serum, saliva, urine, and interstitial fluid). An indicator reagent system and an analyte in a sample of body fluid are reacted to produce a chromatic reaction—the reaction between the reagent and analyte causes the sample to change color. The degree of color change is indicative of the analyte concentration in the body fluid. The color change of the sample is evaluated, for example, using spectroscopy to measure the absorbance level of the transmitted light. Regular transmission spectroscopy is described in detail in U.S. Pat. No. 5,866,349. Diffuse reflectance and fluorescence spectroscopy is described in detail in U.S. Pat. No. 5,518,689 (entitled “Diffuse Light Reflectance Readhead”); U.S. Pat. No. 5,611,999 (entitled “Diffuse Light Reflectance Readhead”); and U.S. Pat. No. 5,194,393 (entitled “Optical Biosensor and Method of Use”).
At a rudimentary level, a transmission spectroscopic analysis includes a light source that produces a beam of light for illuminating a sample and a detector for detecting light that is transmitted through the sample. The detected transmitted light is then compared to a reference sample (e.g., light from the source directly detected by the detector without the sample present). Regular transmission spectroscopy refers to the collection and analysis of the light that exits the sample at small angles (e.g., from about 0° to about) 15° relative to the normal optical axis, and not the scattered light transmitted through the sample. The normal optical axis is an axis that is perpendicular to the sample cell optical entrance and exit widows. Total transmission spectroscopy refers to the collection of substantially all of the light (including scattered light) exiting a sample at large angles (e.g., from about 0° to about) 90° relative to the normal optical axis. Existing systems for total transmission spectroscopic analysis implement an integrating sphere for collecting all of the light passing through the sample, and a required photomultiplier tube for reading the reflected light from a small portion of the inside surface of the integrating sphere.
As reported in an article entitled “Data Preprocessing and Partial Least Squares Analysis for Reagentless Determination of Hemoglobin Concentration Using Conventional and Total Transmission Spectroscopy,” which appeared in the April 2001 of the Journal of Biomedical Optics (Vol. 6, No. 2), regular transmission levels (scatter excluded) of whole blood has hemoglobin concentrations ranging from about 6.6 to 17.2 g/dL are 15.8 to 0.1% T throughout the visible and near-infrared range (e.g., about 500 nm to about 800 nm) with a pathlength of only 100 μm; but, total transmission levels (scatter included) of whole blood has hemoglobin concentrations within the same range are 79% T to 2% T. The total transmission of light having a wavelength ranging from about 600 nm to about 800 nm is nearly 100% T, and there is little separation between the different hemoglobin levels. Thus, the hemoglobin concentration level has little impact on the transmitted light having a wavelength ranging from about 600 nm to about 800 nm.
A drawback associated with existing total transmission spectroscopy systems that use an integrating sphere is a low signal level that requires using a photomultiplier tube for reading the reflected light from a small portion of the inside surface of the integrating sphere. Another drawback associated with conventional total transmission spectroscopy systems is the cost of an integrating sphere and photomultiplier tube. The cost of these devices makes it cost-prohibitive to produce existing total transmission spectroscopy systems for use by a patient needing to self-test, for example, the patient's blood-glucose concentration level. As a result, spectroscopic systems for use in determining the analyte concentration in body fluids have centered on regular transmission measurements.
Existing systems using regular transmission spectroscopy also have several drawbacks. As discussed above, only the light emerging from the sample at small angles is collected using existing regular transmission spectroscopy measurements, often resulting in losing light exiting the sample at large angles. A significant portion of light scattered by the red blood cells is not collected with existing systems using regular transmission measurements, which can lead to significant loss of light resulting in very low transmission levels through whole blood.
To reduce the transmission losses using existing regular transmission systems, a reagent or detergent is typically added to the blood sample to lyse the red blood cells. Rupture of the cell walls through lysis of the blood cells reduces the scattered transmission, and increases the regular transmission of light through the sample. The addition of a lysing reagent and subsequent lysis of the red blood cells is time consuming relative to the overall measurement process. This problem is not present in existing total transmission spectroscopy methods because the scattered transmitted light and regular transmitted light is collected by the optics. Total transmission levels are typically high enough that lysing the red blood cells is not required, which significantly reduces the overall time for a chemical assay.
Another drawback associated with existing systems using regular transmission spectroscopy is a transmission bias at wavelengths of light where the chromatic reaction occurs. The indicator reagent may react with intracellular components (i.e., hemoglobin, lactate dehydogenase, etc.) released from the lysing of red blood cells causing an additional color response. The transmission bias caused by this reaction of the reagent and the certain intracellular components such as hemoglobin is not indicative of the blood-glucose level. This transmission bias causes inaccuracies in determining the analyte (e.g., glucose) concentration. The amount of bias is related to the concentration of certain cellular components in the blood cells.
Since blood lysis is not required for existing total transmission spectroscopy methods, the amount of intracellular components that may interfere with the glucose measurement is significantly reduced. Bias, however, remains for substances such as hemoglobin that absorb at visible wavelengths less than about 600 nm. It is known from the aforementioned article in the Journal of Biomedical Optics, for example, that total transmission spectra of oxy-hemoglobin has absorbance peaks at wavelengths of about 542 nm and about 577 nm. It is known that the absorbance level at wavelengths of about 542 nm or about 577 nm may be used to determine the hemoglobin concentration of the whole blood sample. The remaining interference error in glucose concentration caused by hemoglobin may be corrected for by measuring the total transmission at 542 nm or 577 nm, and correlating the absorption to known hemoglobin concentration.
The hematocrit level of whole blood may also cause a total transmission bias due to differences in the amount of scattered light at different hematocrit levels. The transmission loss caused by varying levels of hematocrit is not indicative of the blood-glucose level. Existing systems using regular transmission or total transmission spectroscopy are not capable of detecting the difference in hematocrit levels because of poor transmission level and poor separation between hematocrit levels at certain wavelengths of light.
Another drawback to existing systems using regular transmission spectroscopy is accuracy errors that result from the sample path length. A 10% variation in the path length of the sample cell area results in a 10% error in the concentration measurement for both regular and total transmission methods. The mechanical tolerance that causes the path length variation is substantially the same regardless of the path length. Existing systems using regular transmission methods, however, require a shorter path length to make up for transmission losses due to red blood cell scatter. Thus, the mechanical tolerance at a shorter pathlength results in higher concentration errors. A longer pathlength—permitted by total transmission spectroscopy systems that collect scattered light from red blood cells—reduces pathlength error.
Therefore, it would be desirable to reduce or eliminate the above described problems encountered by existing systems using regular or total transmission spectroscopy in determining analyte concentration in body fluid.